Carbohydrate Metabolism

(1)

Carbohydrate

Metabolism

8

CHAPTER

OUTLINE

METABOLISM AND JET ENGINES 8.1 GLYCOLYSIS

The Reactions of the Glycolytic Pathway

The Fates of Pyruvate The Energetics of Glycolysis Regulation of Glycolysis

8.2 GLUCONEOGENESIS

Gluconeogenesis Reactions Gluconeogenesis Substrates Gluconeogenesis Regulation

8.3 THE PENTOSE PHOSPHATE PATHWAY 8.4 METABOLISM OF OTHER IMPORTANT SUGARS

Fructose Metabolism

8.5 GLYCOGEN METABOLISM

Glycogenesis Glycogenolysis

Regulation of Glycogen Metabolism

BIOCHEMISTRY INPERSPECTIVE

Saccharomyces Cerevisiae and the Crabtree Effect

BIOCHEMISTRY INPERSPECTIVE Turbo Design Can Be Dangerous Available online

BIOCHEMISTRY INPERSPECTIVE Fermentation: An Ancient Heritage

Wine: A Product of Fermentation

Humans use microorganisms, in this case yeast, to metabolize sugar in the absence of oxygen. The aging of wine in oak barrels improves its taste and aroma.

(2)

Metabolism and Jet Engines

the end of the pathway) to run thermodynamically downhill. As a result, the pathway can produce ATP under varying substrate and product concentra-tions. The term “turbo design,” inspired by the turbo engines in jet aircraft, describes this phenomenon. A good example is glycolysis, the energy-capturing reaction pathway that converts the hexose glucose into pyruvate (Figure 8.1).

A jet engine creates propulsion by mixing air with fuel to create hot, expanding, fast-moving exhaust gases, which blast out the back. Some of the air drawn in at the front of the engine is diverted into compressors, where its pressure increases substantially before flowing into combustion cham-bers and mixing with fuel molecules. As the burning fuel molecules expand, they flow through turbines fitted with fan blades that drive the compressors. An important feature of this process is that hot exhaust gases are also fed back into the engine to accelerate the fuel input step. This chapter reveals how carbohydrate fuel is burned by living cells with the same remarkable efficiency.

C

an systems biology improve our understand-ing of biochemical pathways such as glycolysis? Modern species are the result of billions of years of rigorous natural selection, which has adapted organisms to their various environments. This selection process also governs the metabolic path-ways that manage the biochemical transformations that sustain life. As systems biologists analyzed metabolic processes, it became apparent that evo-lution, operating under thermodynamic and kinetic constraints, has converged again and again into a relatively small set of designs. Catabolic pathways, those that degrade organic molecules and release energy, provide an important example. These path-ways typically have two characteristics: optimal ATP production and kinetic efficiency (i.e., minimal response time to changes in cellular metabolic requirements). Living organisms have optimized catabolic pathways, in part, through highly exer-gonic reactions at the beginning of a pathway. The early “activation” of nutrient molecules thus makes subsequent ATP-producing reactions (usually near

Glucose Compressor Turbo engine Exhaust Fuel

+

ATP 2 ATP 2 ATP 2 ADP Glyceraldehyde-3-phosphate Stage 1 (a) (b) Stage 2 Pyruvate FIGURE 8.1

Comparison of Glycolysis and the Turbo Jet Engine

(a) Glycolysis is a two-stage catabolic pathway. Two of the four ATPs produced in stage 2 are used to activate an incoming glucose molecule (stage 1). The ADPs used in stage 2 are generated from the two ATPs used in stage 1 and in ATP-requiring reactions throughout the cell. (b) The schematic representation of a turbo jet engine illus-trates how energy generated by an engine can be used to improve efficiency. Before exiting the engine, hot exhaust gases are diverted around the compressor turbines, raising the temperature and increasing the efficiency of fuel combustion.

(3)

8.1 Glycolysis 3

Overview

CARBOHYDRATES PLAY SEVERAL CRUCIAL ROLES IN THE METABOLIC PROCESSES OF LIVING ORGANISMS. THEY SERVE AS ENERGY SOURCES AND as structural elements in living cells. This chapter looks at the role of carbohydrates in energy production. Because the monosaccharide glucose is a prominent energy source in almost all living cells, major emphasis is placed on its synthesis, degra-dation, and storage.

L

iving cells are in a state of ceaseless activity. To maintain its “life,” each cell depends on highly coordinated biochemical reactions. Carbohydrates are an important source of the energy that drives these reactions. This chapter discusses the energy-generating pathways of carbohydrate metabolism are discussed. During glycolysis, an ancient pathway found in almost all organisms, a small amount of energy is captured as a glucose molecule is converted to two molecules of pyruvate. Glycogen, a storage form of glucose in vertebrates, is synthesized by glycogene-sis when glucose levels are high and degraded by glycogenolyglycogene-sis when glucose is in short supply. Glucose can also be synthesized from noncarbohydrate precur-sors by reactions referred to as gluconeogenesis. The pentose phosphate pathway enables cells to convert glucose-6-phosphate, a derivative of glucose, to ribose-5-phosphate (the sugar used to synthesize nucleotides and nucleic acids) and other types of monosaccharides. NADPH, an important cellular reducing agent, is also produced by this pathway. In Chapter 9, the glyoxylate cycle, used by some organisms (primarily plants) to manufacture carbohydrate from fatty acids, is considered. Photosynthesis, a process in which light energy is captured to drive carbohydrate synthesis, is described in Chapter 13.

Any discussion of carbohydrate metabolism focuses on the synthesis and usage of glucose, a major fuel for most organisms. In vertebrates, glucose is transported throughout the body in the blood. If cellular energy reserves are low, glucose is degraded by the glycolytic pathway. Glucose molecules not required for immedi-ate energy production are stored as glycogen in liver and muscle. The energy requirements of many tissues (e.g., brain, red blood cells, and exercising skeletal muscle cells) depend on an uninterrupted flow of glucose. Depending on a cell’s metabolic requirements, glucose can also be used to synthesize, for example, other monosaccharides, fatty acids, and certain amino acids. Figure 8.2 summarizes the major pathways of carbohydrate metabolism in animals.

8.1 GLYCOLYSIS

Glycolysis, occurs, at least in part, in almost every living cell. This series of reactions is believed to be among the oldest of all the biochemical pathways. Both the enzymes and the number and mechanisms of the steps in the pathway are highly conserved in prokaryotes and eukaryotes. Also, glycolysis is an anaero-bic process, which would have been necessary in the oxygen-poor atmosphere of pre-eukaryotic Earth.

In glycolysis, also referred to as the Embden-Meyerhof-Parnas pathway, each glucose molecule is split and converted to two three-carbon units (pyruvate). During this process several carbon atoms are oxidized. The small amount of energy captured during glycolytic reactions (about 5% of the total available) is stored temporarily in two molecules each of ATP and NADH (the reduced form of the coenzyme NAD+_{). The subsequent metabolic fate of pyruvate depends on the} organism being considered and its metabolic circumstances. In anaerobic organisms

(4)

4 CHAPTER EIGHT Carbohydrate Metabolism Gluconeogenesis Fatty acids Glycogen Glucose Pyruvate Glycogenesis Glycogenolysis Glycolysis Lactate Citric acid cycle Electron transport system Certain amino acids CO₂ + H₂O + pathway Pentose phosphate Pentose and other sugars Acetyl-CoA ATP FIGURE 8.2

Major Pathways in Carbohydrate Metabolism

In animals, excess glucose is converted to its storage form, glycogen, by glycogenesis. When glucose is needed as a source of energy or as a precursor molecule in biosynthetic processes, glycogen is degraded by glycogenolysis. Glucose can be converted to ribose-5-phosphate (a component of nucleotides) and NADPH (a powerful reducing agent) by means of the pentose phosphate pathway. Glucose is oxidized by glycolysis, an energy-generating pathway that converts it to pyruvate. In the absence of oxygen, pyruvate is converted to lactate. When oxy-gen is present, pyruvate is further degraded to form acetyl-CoA. Significant amounts of energy in the form of ATP can be extracted from acetyl-CoA by the citric acid cycle and the electron transport system. Note that carbohydrate metabolism is inextricably linked to the metabolism of other nutrients. For example, acetyl-CoA is also generated from the break-down of fatty acids and certain amino acids. When acetyl-CoA is

present in excess, a different pathway converts it into fatty acids.

(those that do not use oxygen to generate energy), pyruvate may be converted to waste products such as ethanol, lactic acid, acetic acid, and similar molecules. Using oxygen as a terminal electron acceptor, aerobic organisms such as animals and plants completely oxidize pyruvate to form CO2and H2O in an elaborate step-wise mechanism known as aerobic respiration (Chapters 9 and 10).

Glycolysis (Figure 8.3), which consists of 10 reactions, occurs in two stages: 1. Glucose is phosphorylated twice and cleaved to form two molecules of glyceraldehyde-3-phosphate (G-3-P). The two ATP molecules consumed during this stage are like an investment, because this stage creates the actual substrates for oxidation in a form that is trapped inside the cell.

2. Glyceraldehyde-3-phosphate is converted to pyruvate. Four ATP and two NADH molecules are produced. Because two ATP were consumed in stage 1, the net production of ATP per glucose molecule is 2.

The glycolytic pathway can be summed up in the following equation: D-Glucose 2 ADP 2 Pi 2 NAD→ 2 pyruvate 2 ATP

2 NADH 2H_2H 2O

(5)

8.1 Glycolysis 5 Glucose -6-phosphate Fructose -6-phosphate Fructose-1,6-bisphosphate Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate Glycerate-1,3-bisphosphate Glycerate-2-phosphate Phosphoenolpyruvate Glycerate-1,3-bisphosphate Glycerate-3-phosphate Glycerate-2-phosphate Phosphoenolpyruvate Glycerate-3-phosphate + H+ P_i H₂O H₂O + H+ P_i STAGE 1 1 2 3 4 4 5 6 7 8 9 10 STAGE 2 ADP ATP Glucose NADH Pyruvate ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP NADH Pyruvate NAD+ NAD+ FIGURE 8.3 The Glycolytic Pathway

In glycolysis, a pathway with 10 reactions, each glucose molecule is converted into two pyruvate molecules. In addi-tion, two molecules each of ATP and NADH are produced. Reactions with double arrows are reversible reactions, and those with single arrows are irreversible reactions that serve as control points in the pathway.

(6)

6 CHAPTER EIGHT Carbohydrate Metabolism

The Reactions of the Glycolytic Pathway

Glycolysis is summarized in Figures 8.3. The 10 reactions of the glycolytic path-way are as follows.

1. Synthesis of glucose-6-phosphate. Immediately after entering a cell, glucose and other sugar molecules are phosphorylated. Phosphorylation prevents transport of glucose out of the cell and increases the reactivity of the oxygen in the resulting phosphate ester. Several enzymes, called the hexokinases, catalyze the phosphorylation of hexoses in all cells in the body. ATP, a cosubstrate in the reaction, is complexed with Mg2. (ATP-Mg2complexes are common in kinase-catalyzed reactions.) Under intracellular conditions the reaction is irreversible; that is, the enzyme has no ability to retain or accommodate the product of the reaction in its active site, regardless of the concentration of G-6-P.

+ Glucose Glucose-6-phosphate HO HO OH O HO CH₂ HO + Hexokinase Mg2+ HO CH₂ HO OH O HO O P –O O O– ATP ADP

2. Conversion of glucose-6-phosphate to fructose-6-phosphate. During reac-tion 2 of glycolysis, the open chain form of the aldose glucose-6-phosphate is converted to the open chain form of the ketose fructose-6-phosphate by phosphoglucose isomerase (PGI) in a readily reversible reaction:

Phosphoglucose isomerase Glucose-6-phosphate Fructose-6-phosphate HO HO O OH CH₂ P –O O O– CH₂ O OH HO HO CH₂ OH O P –O O O– HO O

Recall that the isomerization reaction of glucose and fructose involves an enediol intermediate (Figure 7.16). This transformation makes C-1 of the fructose product available for phosphorylation. The hemiacetal hydroxy group of glucose-6-phosphate is more difficult to phosphorylate.

3. The phosphorylation of fructose-6-phosphate. Phosphofructokinase-1 (PFK-1) irreversibly catalyzes the phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate: + PFK-1 Mg2+ HO HO O OH CH₂ P –O O O– CH₂ Fructose-6-phosphate + O OH HO HO O OH CH₂ P –O O O– CH₂ P O− O O− Fructose-1,6-bisphosphate O O ADP ATP

(7)

8.1 Glycolysis 7

The PFK-1-catalyzed reaction is irreversible under cellular conditions. It is, therefore, the first committed step in glycolysis. Unlike glucose-6-phosphate and fructose-6-glucose-6-phosphate, the substrate and product, respec-tively, of the previous reaction, fructose-1,6-bisphosphate cannot be diverted into other pathways. Investing a second molecule of ATP serves several purposes. First of all, because ATP is used as the phosphorylating agent, the reaction proceeds with a large decrease in free energy. After fruc-tose-1,6-bisphosphate has been synthesized, the cell is committed to gly-colysis. Because fructose-1,6-bisphosphate eventually splits into two trioses, another purpose for phosphorylation is to prevent any later product from diffusing out of the cell because charged molecules cannot easily cross membranes.

4. Cleavage of fructose-1,6-bisphosphate. Stage 1 of glycolysis ends with the cleavage of fructose-1,6-bisphosphate into two three-carbon mole-cules: glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHAP). This reaction is an aldol cleavage, hence the name of the enzyme: aldolase. Aldol cleavages are the reverse of aldol condensa-tions, described on p. xxx. In aldol cleavages an aldehyde and a ketone are products. HO HO O OH 6 CH₂ P −O O O− 1 CH₂ + Fructose-1,6-bisphosphate Dihydroxyacetone phosphate 2 3 4 5 P O− O O− O 1 CH₂ HO 2 C 3 CH₂ P O− O O− O O Aldolase O HO 5C 6 CH₂ O H 4 C H P O− O O− O Glyceraldehyde-3-phosphate

Although the cleavage of fructose-1,6-bisphosphate is thermodynamically unfavorable (G 23.8 kJ/mol), the reaction proceeds because the products are rapidly removed.

5. The interconversion of glyceraldehyde-3-phosphate and dihydroxy-acetone phosphate. Of the two products of the aldolase reaction, only G-3-P serves as a substrate for the next reaction in glycolysis. To pre-vent the loss of the other three-carbon unit from the glycolytic pathway, triose phosphate isomerase catalyzes the reversible conversion of DHAP to G-3-P:

After this reaction, the original molecule of glucose has been converted to two molecules of G-3-P.

6. Oxidation of glyceraldehyde-3-phosphate. During reaction 6 of glycolysis, G-3-P undergoes oxidation and phosphorylation. The product,

HO C CH₂ O H C H P O− O O− O Glyceraldehyde-3-phosphate Triose phosphate isomerase HO C CH₂ O CH₂ P O− O O− O Dihydroxyacetone phosphate

(8)

glycerate-1,3-bisphosphate, contains a high-energy phosphoanhydride bond, which may be used in the next reaction to generate ATP:

This complex process is catalyzed by glyceraldehyde-3-phosphate dehy-drogenase, a tetramer composed of four identical subunits. Each subunit contains one binding site for G-3-P and another for NAD, an oxidizing ogent. As the enzyme forms a covalent thioester bond with the substrate (Figure 8.4), a hydride ion (H:) is transferred to NADin the active site. NADH, the reduced form of NAD, then leaves the active site and is replaced by an incoming NAD. The acyl enzyme adduct is attacked by inorganic phosphate and the product leaves the active site.

7. Phosphoryl group transfer. In this reaction ATP is synthesized as phos-phoglycerate kinase catalyzes the transfer of the high-energy phosphoryl group of glycerate-1,3-bisphosphate to ADP:

Reaction 7 is an example of a substrate-level phosphorylation. Because the synthesis of ATP is endergonic, it requires an energy source. In substrate-level phosphorylations, ATP is produced by the transfer of a phosphoryl group from a substrate with a high phosphoryl transfer potential (glycer-ate-1,3-bisphosphate) (refer to Table 4.1) to produce a compound with a lower transfer potential (ATP) and therefore G 0. Because two mole-cules of glycerate-1,3-bisphosphate are formed for every glucose molecule, this reaction produces two ATP molecules, and the investment of phosphate bond energy is recovered. ATP synthesis later in the pathway represents a net gain.

8. The interconversion of 3-phosphoglycerate and 2-phosphoglycerate. Glycerate-3-phosphate has a low phosphoryl group transfer potential. As such, it is a poor candidate for further ATP synthesis (G for ATP synthesis is –30.5 kJ/mol). Cells convert glycerate-3-phosphate with its energy-poor phosphate ester to phosphoenolpyruvate (PEP), which has an exception-ally high phosphoryl group transfer potential. (The standard free energies of hydrolysis of glycerate-3- phosphate and PEP are 12.6 and 61.9 kJ/mol, respectively.) In the first step in this conversion (reaction 8), phos-phoglycerate mutase catalyzes the conversion of a C-3 phosphorylated compound to a C-2 phosphorylated compound through a two-step addition/elimination cycle. H+ P_i Glycerate-1,3-bisphosphate Glyceraldehyde-3-phosphate + + + + O− C HO C CH₂ O H H P O− O O Glyceraldehyde-3-phosphate dehydrogenase H C HO CH₂ P O− O O− O C P O− O O− O O NADH NAD+ + + Phosphoglycerate kinase Mg2+ Glycerate-3-phosphate HO C CH₂ P O − O O− O C O− O H Glycerate-1,3-bisphosphate HO C CH₂ P O− O O− O C P O − O O− O O H ADP ATP

(9)

8.1 Glycolysis 9 +B N H H S C O C C OH H O H H P O− O O− H NAD+ H + C H₂N O Enzyme C OH H O− H C H O− O P O− O C O P O− O O Glycerate-1,3-bisphosphate 3. 4. 1. 2. N SH Enzyme H B: NAD+ C H₂N O H S H C O C C OH H O H H P O− O O− B: H Enzyme NAD+ Glyceraldehyde-3-phosphate C H₂N O S C O C C OH H H H Enzyme +B H O P O− O O− −O P O− O OH N H NAD+ + C H₂N O

..

N H H NADH + H+ C O H₂N S C O C C OH H H H Enzyme +B H O P O− O O− N + Glyceraldehyde-3-phosphate 5. NADH NAD+ + + H+ FIGURE 8.4

Glyceraldehyde-3-Phosphate Dehydrogenase Reaction

In the first step the substrate, glyceraldehyde-3-phosphate, enters the active site. As the enzyme catalyzes the reaction of the substrate with a sulfhydryl group within the active site (step 2), the substrate is oxidized (step 3). The noncovalently bound NADH is exchanged for a cyto-plasmic NAD(step 4). Displacement of the enzyme by inorganic phosphate (step 5) liberates the product, glycerate-1, 3-bisphosphate, thus returning the enzyme to its original form.

(10)

9. Dehydration of 2-phosphoglycerate. Enolase catalyzes the dehydra-tion of glycerate-2-phosphate to form PEP:

PEP has a higher phosphoryl group transfer potential than does glycerate-2- phosphate because it contains an enol-phosphate group instead of a sim-ple phosphate ester. The reason for this difference is made apparent in the next reaction. Aldehydes and ketones have two isomeric forms. The enol form contains a carbon-carbon double bond and a hydroxyl group. Enols exist in equilibrium with the more stable carbonyl-containing keto form. The interconversion of keto and enol forms, also called tautomers, is referred to as tautomerization:

This tautomerization is restricted by the presence of the phosphate group, as is the resonance stabilization of the free phosphate ion. As a result, phosphoryl transfer to ADP in reaction 10 is highly favored.

10. Synthesis of pyruvate. In the final reaction of glycolysis, pyruvate kinase catalyzes the transfer of a phosphoryl group from PEP to ADP. Two mol-ecules of ATP are formed for each molecule of glucose.

H₂O P_i P_i Phosphoglycerate mutase HO C CH₂ P O− O + O− O Glycerate-3-phosphate C O− O H HO C CH₂ P O− O O− O Glycerate-2-phosphate C O− O H C CH₂ P O− O O− O C O− O H P O− O O− O Phosphoglycerate mutase Glycerate-2,3-Bisphosphate Enolase C CH₂OH P O− O O− O Glycerate-2-phosphate C O− O H C CH₂ P O− O O− O Phosphoenolpyruvate (PEP) C O− O + H₂O H+

PEP Pyruvate (enol form) Pyruvate (keto form)

C CH₃ O C O− O + CH₂ P O− O O− O C O− O C CH₂ O C O− C O− ADP ATP Mg2+

(11)

8.1 Glycolysis 11 Hexokinase Phosphoglucoisomerase PKF-1 Aldolase Triose phosphate isomerase Glucose HO HO OH O CH₂ HO HO Glucose-6-phosphate HO CH₂ HO OH O HO O P –O O O– Fructose-1,6-bisphosphate P O O O– O– CH₂ Fructose-6-phosphate HO HO O OH CH₂ P –O O O– CH₂ O OH HO HO O OH CH₂ P –O O O– O Dihydroxyacetone phosphate CH₂ HO C CH₂ P O− O O− O O C HO CH₂ O H C H P O− O O− O Glyceraldehyde-3-phosphate ATP ATP ADP ADP 1 (a) 2 3 4 5 Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate mutase Phosphoglycerate kinase HO C CH₂ O H C H P O– O O– O HO C CH₂ O H C O– P O– O O– O C CH₂ OH O H C O– P O– O O– O H C HO CH₂ P O– O O– O C P O– O O− O O 6 (b) 7 8 9 10 Pi H+ NAD+ NADH + + ATP ADP Glycerate-2-phosphate Phosphoenolpyruvate (PEP) Glycerate-3-phosphate Glycerate-1,3-bisphosphate Pyruvate kinase Pyruvate O C CH₃ O C O– ATP ADP Enolase H2O O CH₂ O O– P O– O O– C C FIGURE 8.5

The Reactions of Glycolysis

In all there are 10 reactions in the glycolytic pathway. (a) In stage 1, reactions 1 through 5 convert glucose into glyceralde-hyde-3-phosphate. Two ATP are consumed in state 1 for each glucose molecule. (b) In stage 2 reactions 6 though 10 convert glyceraldehyde-3-phosphate into pyruvate. In addition to pyruvate, the reactions of stage 2 also produce 4 ATP and 2 NADH per glucose molecule.

(12)

12 CHAPTER EIGHT Carbohydrate Metabolism OH Pyruvate C CH₃ O C O− O + + CH₃ O C HO H C O− H₂O NADH NADH NADH CO₂ CO₂ CH₃CH₂ Lactate Ethanol

Citric acid cycle and electron transport Homolactic fermentation Alcoholic fermentation + + H+ NAD+ NAD+ NAD+ H+ + H+ FIGURE 8.6 The Fates of Pyruvate

When oxygen is available (left), aerobic organisms completely oxidize pyruvate to CO2and H2O. In the absence

of oxygen, pyruvate can be converted to several types of reduced molecules. In some cells (e.g., yeast), ethanol and CO2are produced (middle). In others (e.g., muscle cells), homolactic fermentation occurs in which lactate is

the only organic product (right). Some microorganisms use heterolactic fermentation reactions (not shown) that produce other acids or alcohols in addition to lactate. In all fermentation processes, the principal purpose is to regenerate NAD+_{so that glycolysis can continue.}

PEP is irreversibly converted to pyruvate because in this reaction—the transfer of a phosphoryl group from a molecule with a high transfer potential to one with a lower transfer potential—there is an exceptionally large free energy loss (refer to Table 4.1). This energy loss is associated with the spontaneous conversion (tautomerization) of the enol form of pyruvate to the more stable keto form. The 10 reactions of glycolysis are illustrated in Figure 8.5.

The Fates of Pyruvate

In terms of energy, the result of glycolysis is the production of two ATPs and two NADHs per molecule of glucose. Pyruvate, the other product of glycolysis, is still an energy-rich molecule, which can yield a substantial amount of ATP. Whether or not further energy can be produced, however, depends on the cell type and the availability of oxygen. Under aerobic conditions, most cells in the body convert pyruvate into acetyl-CoA, the entry-level substrate for the citric acid cycle, an amphibolic pathway that completely oxidizes the two acetyl carbons to form CO2and and the reduced molecules NADH and FADH2. (An amphibolic pathway functions in both anabolic and catabolic processes.) The electron trans-port system, a series of oxidation- reduction reactions, transfers electrons from NADH and FADH2to O2to form water. The energy that is released during elec-tron transport is coupled to a mechanism that synthesizes ATP. Under anaerobic conditions, further oxidation of pyruvate is impeded. A number of cells and organ-isms compensate by converting this molecule to a more reduced organic compound and regenerating the NADrequired for glycolysis to continue (Figure 8.6). (Recall

(13)

8.1 Glycolysis 13 H+ _C CH₃ H C O− O Lactate + Pyruvate C CH₃ O C O− O + HO + Lactate dehydrogenase NADH NAD+ + Glycerate-1,3-bisphosphate Glyceraldehyde-3-phosphate Pyruvate Lactate H+ P_i NADH NAD+ FIGURE 8.7

Recycling of NADH During Anaerobic Glycolysis

The NADH produced during the conversion of glyceraldehyde-3-phosphate to glycerate-1,3-bisphosphate is oxidized when pyruvate is converted to lactate. This process allows the cell to continue producing ATP under anaerobic conditions as long as glucose is available.

In rapidly contracting muscle cells, the demand for energy is high. After the O2 supply is depleted, lactic acid fermentation provides sufficient NAD⫹to allow glycolysis (with its low level of ATP production) to continue for a short time (Figure 8.7).

In yeast and certain bacterial species, pyruvate is decarboxylated to form acetaldehyde, which is then reduced by NADH to form ethanol. (In a decar-boxylation reaction, an organic acid loses a carboxyl group as CO2.)

that the hydride ion acceptor molecule NAD+ _{is a cosubstrate in the reactoin catalyzed} by glyceraldehyde-3-phosphate dehydrogenase.) This process of NAD⫹ regenera-tion is referred to as fermentaregenera-tion. Muscle cells, red blood cells, and certain bac-terial species (e.g., Lactobacillus) produce NAD⫹by transforming pyruvate into lactate: Pyruvate Pyruvate decarboxylase CO₂ Acetaldehyde H C CH₃ O C CH₃ O C O− O Ethanol CH₃ OH CH 2 Alcohol dehydrogenase NADH NAD+ + H+

Most molecules of ethanol are detoxified in the liver by two reactions. In the first, ethanol is oxidized to form acetaldehyde. This reaction, catalyzed by alco-hol dehydrogenase, produces large amounts of NADH:

Soon after its production, acetaldehyde is converted to acetate by aldehyde dehy-drogenase, which catalyzes a reaction that also produces NADH:

One common effect of alcohol intoxication is the accumulation of lactate in the blood. Can you explain why this effect occurs?

dehydrogenase Aldehyde C H O CH₃ + NAD+ + H₂O H+ C O O + + CH NADH 3 − 2 H+ C H O + OH CH₂

CH₃ + NAD+ ADH CH₃ NADH +

(14)

Visit the companion website at www.oup.com/us/mckee to read the Biochemistry in Perspective essay on fermentation.

KEY CONCEPTS

• During glycolysis, glucose is converted to two molecules of pyruvate. A small amount of energy is captured in two molecules each of ATP and NADH.

• In anaerobic organisms, pyruvate is con-verted to waste products in a process called fermentation.

• In the presence of oxygen the cells of aerobic organisms convert pyruvate into CO₂and H2O.

This process, called alcoholic fermentation, is used commercially to produce wine, beer, and bread. Certain bacterial species produce organic molecules other than ethanol. For example, Clostridium acetobutylicum, an organism related to the causative agents of botulism and tetanus, produces butanol. Until recently, this organism was used commercially to synthesize butanol, an alcohol used to pro-duce detergents and synthetic fibers. A petroleum-based synthetic process has now replaced microbial fermentation.

The Energetics of Glycolysis

During glycolysis, the energy released as glucose is broken down to pyruvate is coupled to the phosphorylation of ADP with a net yield of 2 ATP. However, eval-uation of the standard free energy changes of the individual reactions (Figure 8.8) does not explain the efficiency of this pathway. A more useful method for evaluating free energy changes takes into account the conditions (e.g., pH and metabolite concentrations) under which cells actually operate. As illustrated in Figure 8.8, free energy changes measured in red blood cells indicate that only three reactions (1, 3, and 10, see pp. 269–270) have significantly negative⌬G values. These reac-tions, catalyzed by hexokinase, PFK-1, and pyruvate kinase, respectively, are for all practical purposes irreversible; that is, each goes to completion as written.

0 GLU G6P F6P FBP GAP GBP PG3 PG2 PEP PYR LAC

+10 0 –10 –20 –30 –40 –50 –60 –70 F ree energy ( k cal) ΔG ⬚

_⬘

ΔG = – 103.8 kJ (– 24.6 kcal) – 130.9 kJ (– 31.3 kcal) ⬚

_⬘

ΔG = ΔG FIGURE 8.8

Free Energy Changes During Glycolysis in Red Blood Cells

Note that the standard free energy changes (⌬G⬚⬘) for the reactions in glycolysis show no consistent pattern (upper plot). In contrast, actual free energy values (⌬G) based on metabolite concentrations measured in red blood cells (lower plot) clearly illustrate why reactions 1, 3, and 10 (the conversions of glucose to glucose-6-phosphate, fruc-tose-6-phosphate to fructose-1,6-bisphosphate, and phosphoenolpyruvate to pyruvate, respectively) are irreversible. The ready reversibility of the remaining reactions is indicated by their near-zero ⌬G values. (GLU = glucose, G6P = glucose-6-phosphate, F6P = fructose-6-phosphate, FBP = fructose-1,6-bisphosphate, GAP = glyceraldehyde phosphate, PG3 = glycerate-3-phosphate, PG2 = glycerate-2-phosphate, PEP = phosphoenolpyruvate, PYR = pyru-vate, LAC = lactate) Note that the conversion of DHAP to GAP is not counted in this list, since FBP is broken into GAP and DHAP, which is reconverted into GAP.

(15)

8.1 Glycolysis 15

Biochemestry

IN PERSPECTIVE

▼

Saccharomyces cerevisiae and the

Crabtree Effect

What unique properties of Saccharomyces cerevisiae

make it so useful in the production of wine, beer, and

bread?

The yeast Saccharomyces cerevisiae, a eukaryotic microorganism, has had a profound effect on humans. Beginning in the Neolithic age and continuing to the present day, this organism is used to con-vert carbohydrate-containing food into the wine, beer, and bread that many humans consider indispensible to life. What proper-ties of S. cerevisiae make it uniquely suited for the oldest of biotechnologies? Although many yeast species can ferment car-bohydrates to form ethanol and CO2, only S. cerevisiae efficiently

produces these molecules in large quantities. A simple experiment helps explain why. If fleshy fruits such as grapes are crushed and placed in a vat, they will begin to ferment. At the beginning of fer-mentation, a survey of the microorganisms present reveals many different types of microorganisms, but relatively few S. cerevisiae. As the fermentation process proceeds (and the ethanol content increases), however, S. cerevisiae cells become a larger proportion of the microbes until eventually they virtually become the only microbes present. How S. cerevisiae performs this feat has been the subject of considerable research—and not only because of the economic importance of traditional fermentation biotech-nologies. The goal of producing ethanol biofuel from cellulose

(not from a food staple such as corn) in an efficient, cost-effec-tive manner still remains elusive. The principal physiological reason that allows S. cerevisiae to ferment carbohydrates effi-ciently and dominate its environment is explained by the Crabtree effect, described next.

The Crabtree Effect

S. cerevisiae is a facultative anaerobe: it is capable of

gener-ating energy in both the presence and the absence of O2 using

aerobic metabolism (the citric acid cycle, the electron transport system, and oxidative phosphorylation) and fermentation, respectively. Unlike most fermenting organisms, S. cerevisiae can also ferment sugar in the presence of O2. As glucose or

fruc-tose levels rise, pyruvate is diverted away from the citric acid cycle (the first phase of aerobic energy generation) into ethanol synthesis by conversion to acetaldehyde and CO2by pyruvate

decarboxylase. This phenomenon, in which glucose represses aerobic metabolism, is the Crabtree effect. (In most organisms that use oxygen the Pasteur effect is observed: glycolysis is depressed when the gas is available.) In S. cerevisiae cells high glucose levels result in changes in gene expression. These changes promote the insertion of hexose transporters into the plasma membrane (resulting in rapid transport of glucose into the cell), the synthesis of glycolytic enzymes, and the inhibition

C6H12O6 2 CH3C O – O Pyruvate Glycolysis NAD+ _NADH ADH1 2CH3CH2OH Ethanol ADH2 PDC 2CO2 2 CH3C H O Acetaldehyde 2 CH3C O– O C OPO32– O 2 CH3 C SCoA O 2 CH3

Acetate Acetyl Phosphate Acetyl CoA

2 ATP 2 ADP 2 CoASH

FIGURE 8A

Ethanol Metabolism in S. cerevisiae

When glucose levels are high, yeast cells shift into the “make, accumulate, con-sume” ethanol pathway. At first, glucose is converted into ethanol molecules to regenerate NAD+_{. Ethanol is then}

released into the environment where it kills competing microbes. Once glucose is depleted, glucose repression ends. Among the results of derepression is that ADH2, the enzyme that converts ethanol back into acetaldehyde, is synthesized. Acetaldehyde is subsequently converted into acetyl-CoA, the substrate for the citric acid cycle. Note that although the “make, accumulate, consume” strategy is expensive (i.e., the energy expended to synthesize the enzymes needed to convert ethanol into acetyl-CoA and the ATPs used to synthesize acetyl phosphate), yeast cells manage to kill off the compe-tition and retrieve a waste product that they then use as an energy source.

(16)

The values for the remaining reactions (2, 4–9) are so close to zero that they oper-ate near equilibrium. Consequently, these latter reactions are easily reversible; small changes in substrate or product concentrations can alter the direction of each reaction. Not surprisingly, in gluconeogenesis (Section 8.2), the pathway by which glucose can be generated from pyruvate and certain other substrates, all of the glycolytic enzymes are involved except for those that catalyze reactions 1, 3, and 10. Gluconeogenesis uses different enzymes to bypass the irreversible steps of glycolysis.

Regulation of Glycolysis

The rate at which the glycolytic pathway operates in a cell is directly controlled primarily by the kinetic properties of its hexokinase isoenzymes and the allosteric regulation of the enzymes that catalyze the three irreversible reactions: hexoki-nase, PFK-1, and pyruvate kinase.

THE HEXOKINASES The animal liver has four hexokinases. Three of these enzymes (hexokinases I, II, and III) are found in varying concentrations in other body tissues, where they bind reversibly to an anion channel (called a porin) in the outer membrane of mitochondria. As a result, ATP is readily available. These isozymes have high affinities for glucose relative to its concentration in blood; that is, they are half- saturated at concentrations of less than 0.1 mM, although blood glucose levels are approximately 4 to 5 mM. In addition, hexokinases I, II, and III are inhibited from phosphorylating glucose molecules by glucose-6-phosphate, the product of the reaction. When blood glucose levels are low, these properties allow cells such as those in brain and muscle to obtain sufficient glucose. When

of aerobic respiration enzyme synthesis. The diversion of pyru-vate into ethanol production is also believed to result from an overflow phenomenon. There are too few pyruvate dehydro-genase molecules to convert pyruvate entirely into acetyl-CoA. Consequently, pyruvate decarboxylase converts excess pyruvate molecules into acetaldehyde.

At first glance the glucose-induced repression of aerobic metabolism appears inefficient because, the production of ATP in fermentation (2 ATP per glucose) is minor compared with that of oxidative phosphorylation (approximately 30 ATP per glucose). However, rapid synthesis of ethanol along with its release into the environment by ethanol-tolerant S. cerevisiae has the effect of eliminating microbial competitors and preda-tors. Once glucose levels are depleted and O2is available, there

is a significant change in gene expression, called the diauxic

shift, which alters yeast energy metabolism. Glucose repression

ends, and the yeast cells proceed to reabsorb ethanol and recon-vert it to acetaldehyde using ADH2 (p. xxx). Acetaldehyde is then converted to acetyl-CoA, the citric acid cycle substrate. In effect, as a result of its unique energy metabolism, referred to as “make, accumulate, consume,” S. cerevisiae cells can use their waste product as an energy source (Figure 8A). Humans take advantage of the first phase of yeast energy metabolism in the commercial production of wine and beer. Early in the process, aerobic fermentation is accompanied by O2-requiring reactions

that facilitate cell division, thereby increasing the number of yeast. As the oxygen in the fermentation vessel is depleted, ethanol production accelerates. Eventually the sugar level drops and fermentation slows. By excluding O2 from the fermentation

at this stage of the process, the ethanol content of the product is maximized because the shift to aerobic degradation of ethanol is prevented.

Biochemistry

IN PERSPECTIVE cont

SUMMARY:

A metabolic adaptation in the ancestors of S. cerevisiae allowed them to produce

large quantities of ethanol, a toxic molecule that eliminated microbial competitors. Humans take advantage of this adaptation when they use S. cerevisiae in the production of alcoholic beverages and bread.

(17)

blood glucose levels are high, cells do not phosphorylate more glucose molecules than required to meet their immediate needs. The fourth enzyme, called hexokinase IV (or glucokinase), catalyzes the same reaction but has significantly different kinetic properties. Gluco kinase (GK), found in liver as well as in certain cells in pancreas, intestine, and brain, requires much higher glucose concentrations for opti-mal activity (about 10 mM), and it is not inhibited by glucose-6-phosphate. In liver, GK diverts glucose into storage as glycogen. This capacity provides the resources used to maintain blood glucose levels, a major role of the liver. Consequently, after a carbohydrate meal the liver does not remove large quantities of glucose from the blood for glycogen synthesis until other tissues have satisfied their require-ments for this molecule. In cell types where it occurs, GK is believed to be a

glu-cose sensor. Because glucokinase does not usually work at maximum velocity, it

is highly sensitive to small changes in blood glucose levels. Its activity is linked to a signal transduction pathway. For example, the release of insulin (the hor-mone that promotes the uptake of glucose into muscle and adipose tissue cells) by pancreatic ␤-cells in response to rising blood levels of glucose is initiated by GK. GK regulation involves its binding to GK regulator protein (GKRP), a process that is triggered by high fructose-6-phosphate levels. GKRP/GK then moves into the nucleus. When blood glucose levels rise after a meal, GKRP releases GK (caused by exchange with fructose-1-phosphate), and GK moves back through the nuclear pores and can again phosphorylate glucose.

ALLOSTERIC REGULATION OF GLYCOLYSIS The reactions catalyzed by hex-okinases I, II, and III, PFK-1, and pyruvate kinase can be switched on and off by allosteric effectors. In general, allosteric effectors are molecules whose cel-lular concentrations are sensitive indicators of a cell’s metabolic state. Some allosteric effectors are product molecules. For example, hexokinases I, II, and III are inhibited by excess glucose-6-phosphate. Several energy-related molecules also act as allosteric effectors. For example, a high AMP concentration (an indi-cator of low energy production) activates pyruvate kinase. In contrast, pyruvate kinase is inhibited by a high ATP concentration (an indicator that the cell’s energy requirements are being met). Acetyl-CoA, which accumulates when ATP is in rich supply, inhibits pyruvate kinase.

Of the three key enzymes in glycolysis, PFK-1 is the most carefully regu-lated. Its activity is allostei cally inhibited by high levels of ATP and citrate, which are indicators that the cell’s energy charge is high and that the citric acid cycle, a major component of the cell’s energy-generating capacity, has slowed down. AMP is an allosteric activator of PFK-1. AMP levels, which increase when the energy charge of the cell is low, are a better predictor of energy deficit than ADP levels. Fructose-2,6-bisphosphate, allosteric activator of PFK-1 activity in the liver, is synthesized by phosphofructokinase-2 (PFK-2) in response to hormonal signals correlated to blood glucose levels (Figure 8.9). When serum glucose levels are high, hormone-stimulated increase in fructose-2,6-bisphosphate coordinately increases the activity of PFK-1 (activates glycolysis) and decreases the activity of the enzyme that catalyzes the reverse reaction, fructose-1,6-bis-phosphatase (inhibits gluconeogenesis, Section 8.2). AMP is an allosteric inhibitor of fructose-1,6-bisphosphatase. PFK-2 is a bifunctional enzyme that behaves as a phosphatase when phosphorylated in response to the hormone glucagon (released into blood in reponse to low blood sugar, see later). It functions as a kinase when dephosphorylated in response to the hormone insulin (high blood sugar). Fructose-2,6-bisphosphate, produced via hormone-induced covalent mod-ification of PFK-2, is an indicator of high levels of available glucose and alloster-ically activates PFK-1. Accumulated fructose-1,6-bisphosphate activates pyruvate kinase, providing a feed-forward mechanism of control (i.e., fructose-1,6-bis-phosphate is an allosteric activator). The allosteric regulation of glycolysis is summarized in Table 8.1.

(18)

HORMONAL REGULATION Glycolysis is also regulated by the peptide hor-mones glucagon and insulin. Glucagon, released by pancreatic ␣-cells when blood glucose is low, activates the phosphatase function of PFK-2, thereby reducing the level of fructose-2,6-bisphosphate in the cell. As a result, PFK-1 activity and flux through glycolysis are decreased. In liver, glucagon also inac-tivates pyruvate kinase. Glucagon’s effects, triggered by binding to its recep-tor on target cell surfaces, are mediated by cyclic AMP. Cyclic AMP (cAMP) (p. xxx) is synthesized from ATP in a reaction catalyzed by adenylate cyclase, a plasma membrane protein. Once synthesized, cAMP binds to and activates protein kinase A (PKA). PKA then initiates a signal cascade of phosphoryla-tion/dephosphorylation reactions that alter the activities of a diverse set of enzymes and transcription factors. Transcription factors are proteins that reg-ulate or initiate RNA synthesis by binding to specific DNA sequences called response elements.

Insulin is a peptide hormone released from pancreatic ␤-cells when blood glucose levels are high. The effects of insulin on glycolysis include activation of the kinase function of PFK-2, which increases the level of fructose-2,6-bisphosphate in the cell, in turn increasing glycolytic flux. In cells containing insulin-sensitive glucose transporters (muscle and adipose tissue but not liver or brain) insulin promotes the translocation of glucose transporters to the cell surface. When insulin binds to its cell-surface receptor, the receptor protein undergoes several autophosphorylation reactions, which trigger numerous intra-cellular signal cascades that involve phosphorylation and dephosphorylation of target enzymes and transcription factors. Many of insulin’s effects on gene expression are mediated by the transcription factor SREBP1c, a sterol regulatory element binding protein (p. xxx). As a result of SREBP1c activation, there is increased synthesis of glucokinase and pyruvate kinase.

AMPK: A METABOLIC MASTER SWITCH AMP-activated protein kinase (AMPK) is an enzyme that plays a central role in energy metabolism. First discov-ered as a regulator of lipid metabolism, AMPK is now known to affect glucose metabolism as well. Once AMPK is activated, as a result of an increase in a

Enzyme Activator Inhibitor

Hexokinase Glucose-6-phosphate, ATP

PFK-1 Fructose-2,6-bisphosphate, AMP Citrate, ATP Pyruvate kinase Fructose-1,6-bisphosphate, AMP Acetyl-CoA, ATP

Allosteric Regulation of Glycolysis

TABLE 8.1 pi Fructose-6-Phosphate FBP-ase-2 Fructose-2,6-bisphosphate Glucagon ATP PKA PPP PFK-2 (Active) PFK-2-PO4 (Inactive) pi Insulin (+) (+) H2O ADP FIGURE 8.9 Fructose-2,6-Bisphosphate Level Regulation

Glycolysis is stimulated when fructose-2,6-bisphosphate, the activator of PFK-1, is synthesized by PFK-2. PFK-2 is, in turn, activated by a dephosphorylation reaction catalyzed by phosphoprotein phosphatase (PPP), an enzyme activated by insulin. PFK-2 is a bifunctional protein with two enzymatic activities: PFK-2 and fructose-2-6-bisphosphatase-2 (FBPase-2). As a result, the dephosphorylation reaction also inhibits FBP-ase-2, the enzymatic activity th at con-verts 2,6-bisphosphate to fructose-6-phosphate. Glycolysis is inhibited when fructose-2-6-bisphosphate levels are low, which is the result of a glucagon-stimulated and protein kinase A (PKA)-catalyzed phos-phorylation reaction that inactivates PFK-2.

(19)

8.2 Gluconeogenesis 19

Insulin is a hormone secreted by the pancreas when blood sugar increases. Its most easily observable function is to reduce the blood sugar level to normal. The binding of insulin to its target cells promotes the transport of glucose across the plasma membrane. The capacity of an individual to respond to a carbohydrate meal by reducing blood glucose concentration quickly is referred to as glucose

tolerance. Chromium-deficient animals show a decreased glucose tolerance; that

is, they cannot remove glucose from blood quickly enough. The metal is believed to facilitate the binding of insulin to cells. Do you think the chromium is acting as an allosteric activator or cofactor?

QUESTION 8.2

Louis Pasteur, the great nineteenth-century French chemist and microbiologist, was the first scientist to observe that cells that can oxidize glucose completely to CO2 and H2O use glucose more rapidly in the absence of O2 than in its presence. The oxygen molecule seems to inhibit glucose consumption. Explain in general terms the significance of this finding, now referred to as the Pasteur effect.

QUESTION 8.3

cell’s AMP:ATP ratio, it phosphorylates target proteins (enzymes and transcrip-tion factors). AMPK switches off anabolic pathways (e.g., protein and lipid syn-thesis) and switches on catabolic pathways (e.g., glycolysis and fatty acid oxidation). AMPK’s regulatory effects on glycolysis include the following. In car-diac and skeletal muscle, AMPK promotes glycolysis by facilitating the stress- or exercise-induced recruitment of glucose transporters to the plasma membrane. In cardiac cells, AMPK stimulates glycolysis by activating PFK-2. AMPK struc-ture and its functional properties are described in Chapter 12.

8.2 GLUCONEOGENESIS

Gluconeogenesis, the formation of new glucose molecules from noncarbohydrate precursors, occurs primarily in the liver. Precursor molecules include lactate, pyru-vate, glycerol, and certain ␣-keto acids (molecules derived from amino acids). Under certain conditions (i.e., metabolic acidosis or starvation) the kidney can make small amounts of new glucose. Between meals adequate blood glucose levels are maintained by the hydrolysis of liver glycogen. When liver glycogen is depleted (e.g., owing to prolonged fasting or vigorous exercise), the gluco-neogenesis pathway provides the body with adequate glucose. Brain and red blood cells rely exclusively on glucose as their energy source.

Gluconeogenesis Reactions

The reaction sequence in gluconeogenesis is largely the reverse of glycolysis. Recall, however, that three glycolytic reactions (the reactions catalyzed by hexokinase, PFK-1, and pyruvate kinase) are irreversible. In gluconeogenesis, alternate reactions catalyzed by different enzymes are used to bypass these obstacles. The reactions unique to gluconeogenesis are listed next. The entire gluconeogenic pathway and its relationship to glycolysis are illustrated in Figure 8.10. The bypass reactions of gluconeogenesis are as follows:

(20)

20 CHAPTER EIGHT Carbohydrate Metabolism Glucose Glycerol + Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate + Glycerate-1,3-bisphosphate Glycerate-3-phosphate P_i Certain amino acids Glycerate-2-phosphate Phosphoenolpyruvate Lactate Pyruvate + + Pyruvate carboxylase Pyruvate kinase PEP carboxykinase Oxaloacetate Certain amino acids + Glucose-1-phosphate

Glycogen UDP- glucose

Glucose-6-phosphate Fructose-1,6-bisphosphate Fructose-6-phosphate Hexokinase Glucose-6-phosphatase PFK-1 Fructose bisphosphate phosphatase H₂O P_i P_i PP_i H₂O H₂O H₂O CO₂ CO₂ P_i ADP ATP UTP NAD+ NAD+ NADH GDP GTP NADH NADH NADH + NAD+ NAD+ ADP ADP ADP ADP ADP ATP ATP ATP ATP ATP H+ H+ H+ H+ FIGURE 8.10

Carbohydrate Metabolism: Gluconeogenesis and Glycolysis

In gluconeogenesis, which occurs when blood sugar levels are low and liver glycogen is depleted, 7 of the 10 reactions of glycolysis are reversed. Three irreversible glycolytic reactions are bypassed by alternative reactions. The major substrates for gluconeogenesis are certain amino acids (derived from muscle), lactate (formed in muscle and red blood cells), and glycerol (produced from the degradation of triacylglycerols). In contrast to the reactions of glycolysis, which occur only in cytoplasm, the gluconeogenesis reactions catalyzed by pyruvate carboxylase and, in some species, PEP carboxykinase occur within the mitochondria. The reaction catalyzed by glucose-6-phosphatase takes place in the endo-plasmic reticulum. Note that gluconeogenesis and glycolysis do not occur simultaneously. In glycolysis, pyruvate is converted either to acetyl-CoA (not shown) or to lactate.

(21)

Turbo Design Can Be Dangerous

Why must turbo design pathways be rigorously

controlled?

Catabolic pathways with a turbo design, such as glycolysis, are optimized and efficient. However, the early phases of such path-ways must be negatively regulated to prevent buildup of inter-mediates and overuse of fuel. Two of the four ATPs produced from each glucose molecule are fed back to the fuel input stage of the pathway to drive the pathway forward. As indicated by its use by most modern living organisms, glycolysis has been a tremendously successful energy-generating strategy. It is not per-fect, however. Under certain circumstances, the turbo design of glycolysis makes some cells vulnerable to a phenomenon called “substrate-accelerated death”.

Certain types of mutant yeast cells, for example, are unable to grow anaerobically on glucose despite having a completely functional glycolytic pathway. These mutants die when exposed to

Biochemistry

IN PERSPECTIVE

large concentrations of glucose. Amazingly, research efforts have revealed that defects in TPS1, the gene that codes for the catalytic subunit of trehalose-6-phosphate synthase, are responsible. Trehalose-6-phosphate (Tre-6-P), an ␣-(1,1)-disaccharide of glu-cose, is a compatible solute (see the Biochemistry in Perspective box in Chapter 3 entitled Water, Abiotic Stress, and Compatible Solutes) used by yeast and various other organisms to resist several forms of abiotic stress. Apparently, Tre-6-P is a normal inhibitor of HK (and possibly a glucose transporter). In the absence of a functional TPS1 protein and when glucose becomes available, gly-colytic flux in the mutant cells rapidly accelerates. In a relatively short time, as a result of the turbo design of the pathway, most available phosphate has been incorporated into glycolytic inter-mediates, and the cell’s ATP level is too low to sustain cellular processes. This and other similar examples of substrate-accelerated cell death in other species provide insight into the importance of the intricate regulatory mechanisms observed in living organisms.

SUMMARY:

Defects in the intricate regulatory mechanism that controls a turbo design pathway

can make an organism vulnerable to substrate-accelerated death through uncontrolled pathway flux.

1. Synthesis of PEP. PEP synthesis from pyruvate requires two enzymes: pyruvate carboxylase and PEP carboxykinase. Pyruvate carboxylase, found within mitochondria, converts pyruvate to oxaloacetate (OAA):

The transfer of CO2 to form the product OAA is mediated by the coenzyme

biotin (p. xxx), which is covalently bound within the enzyme’s active site.

Pyruvate C CH₃ O C O− O Oxaloacetate (OAA) C CH₂ O C O O− C O− O + + + H+ Pyruvate carboxylase (biotin) + P_i CO₂ H₂O ADP ATP

(22)

OAA is then decarboxylated and phosphorylated by PEP car boxykinase in a reaction driven by the hydrolysis of guanosine triphosphate (GTP):

PEP carboxykinase is found within the mitochondria of some species and in the cytoplasm of others. In humans this enzymatic activity is found in both compartments. Because the inner mitochondrial membrane is impermeable to OAA, cells that lack mitochondrial PEP carboxykinase transfer OAA into the cytoplasm by using the malate shuttle. In this process, OAA is converted into malate by mitochondrial malate dehydro-genase. After the transport of malate across mitochondrial membrane, the reverse reaction (to form OAA and NADH) is catalyzed by cytoplasmic malate dehydrogenase. The malate shuttle allows gluconeogenesis to con-tinue because it provides the NADH required for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

2. Conversion of fructose-1,6-bisphosphate to fructose-6-phosphate. The irreversible PFK-1–catalyzed reaction in glycolysis is bypassed by fructose-1,6-bisphosphatase:

This exergonic reaction (G –16.7 kJ/mol) is also irreversible under cellular conditions. ATP is not regenerated, and inorganic phosphate (Pi) is also produced. Fructose-1,6-bisphosphatase is an allosteric enzyme. Its activity is stimulated by citrate and inhibited by AMP and fructose-2,6-bisphosphate. + Malate dehydrogenase OAA H+ + + Malate CH₂ C O O− C O− C O O C CH₂ H C O O− C O− HO O NAD+ NADH + OAA C CH₂ O C O O− C O− O PEP C CH₂ O C O− O P O− O− O PEP carboxykinase GTP GDP CO2 Fructose-1,6-bisphosphatase + HO HO O OH CH₂ P −O O O− CH₂ P O− O O− Fructose-1,6-bisphosphate P_i Fructose-6-phosphate HO HO O OH CH₂ P −O O O− CH₂OH O O O + H₂O

(23)

Patients with von Gierke’s disease (a glycogen storage disease) lack glucose-6-phosphatase activity. Two prominent symptoms of this disorder are fasting hypo-glycemia and lactic acidosis. Can you explain why these symptoms occur?

QUESTION 8.6 H + HPO ₄ 2C₃H ₄O ₃ + 4 + 2 + 2 + 2 + 1C₆H ₁₂O₆ + 4 + 2 + 2 + 6 + 6 H + Glucose Pyruvic acid 2 − 6 H ₂O ADP A TP GDP GTP NAD + NADH

3. Formation of glucose from glucose-6-phosphate. Glucose-6-phosphatase, found only in liver and kidney, catalyzes the irreversible hydrolysis of glu-cose-6-phosphate to form glucose and Pi. Glucose is subsequently released into the blood.

Each of the foregoing reactions is matched by an opposing irreversible reaction in glycolysis. Each set of such paired reactions is referred to as a substrate cycle. Because they are coordinately regulated (an activator of the enzyme catalyzing the forward reaction serves as an inhibitor of the enzyme catalyzing the reverse reaction), very little energy is wasted, even though both enzymes may be operating at some level at the same time.

Flux control (regulation of the flow of substrate and removal of product)

is more effective if transient accumulation of product is funneled back through the cycle. The catalytic velocity of the forward enzyme will remain high if the concentration of the substrate is maximized. The gain in catalytic efficiency more than makes up for the small energy loss in recycling the product.

Gluconeogenesis is an energy-consuming process. Instead of generating ATP (as in glycolysis), gluconeogenesis requires the hydrolysis of six high-energy phosphate bonds.

Malignant hyperthermia is a rare, inherited disorder triggered during surgery

by certain anesthetics. A dramatic (and dangerous) rise in body temperature (as high as 112F) is accompanied by muscle rigidity and acidosis. The excessive muscle contraction is initiated by a large release of calcium from the sarcoplas-mic reticulum, a calcium-storing organelle in muscle cells. Acidosis results from excessive lactic acid production. Prompt treatment to reduce body temperature and to counteract the acidosis is essential to save the patient’s life. A probable contributing factor to this disorder is wasteful cycling between glycolysis and glu-coneogenesis. Explain why this is a reasonable explanation.

QUESTION 8.4

After examining the gluconeogenic pathway reaction summary illustrated here, account for each component in the equation. [Hint: The hydrolysis of each nucleotide releases a proton.]

QUESTION 8.5

Malignant Hyperthermia

(24)

24 CHAPTER EIGHT Carbohydrate Metabolism FIGURE 8.11

The Cori Cycle

During strenuous exercise, lactate is produced anaerobically in muscle cells. After passing through blood to the liver, lactate is converted to glucose by gluconeogenesis. C CH₂ O O− O CH₂OH P O O− DHAP Glycerol C CH₂OH H OH CH₂OH Glycerol-3-phosphate C H OH CH₂OH CH₂ O P O− O O− Glycerol kinase ADP ATP + H+ Glycerol phosphate dehydrogenase NAD+ NADH Bloodstream Glucose Glucose Pyruvate Lactate Glucose-6-phosphate Pyruvate Lactate Lactate Glucose

Gluconeogenesis Substrates

As previously mentioned, several metabolites are gluconeogenic precursors. Three of the most important substrates are described briefly.

Lactate is released by red blood cells and other cells that lack mitochondria or have low oxygen concentrations. In the Cori cycle, lactate is released by skeletal mus-cle during exercise (Figure 8.11). After lactate is transferred to the liver, it is recon-verted to pyruvate by lactate dehydrogenase and then to glucose by gluconeogenesis. Glycerol, a product of fat metabolism in adipose tissue, is transported to the liver in the blood and then converted to glycerol-3-phosphate by glycerol kinase. Oxidation of glycerol-3-phosphate to form DHAP occurs when cytoplasm NAD concentration is relatively high.

(25)

8.2 Gluconeogenesis 25 Glycolosis Bloodstream Gluconeogenesis Urea Urea cycle Alanine Glucose Glucose Glucose α-Ketoglutarate α-Ketoglutarate Alanine

transaminase _transaminaseAlanine

Glutamate _Glutamate NH3 _NH Amino acids Muscle protein Alanine Alanine Pyruvate Pyruvate 3 FIGURE 8.12

The Glucose-Alanine Cycle

Alanine is formed from pyruvate in muscle. After it has been transported to the liver, alanine is reconverted to pyru-vate by alanine transaminase. Eventually pyrupyru-vate is used in the synthesis of new glucose. Because muscle cannot synthesize urea from amino nitrogen, the glucose-alanine cycle is used to transfer amino nitrogen to the liver.

Of all the amino acids that can be converted to glycolytic intermediates (mol-ecules referred to as glucogenic), alanine is perhaps the most important. When exercising muscle produces large quantities of pyruvate, some of these molecules are converted to alanine by a transamination reaction involving glutamate:

After it has been transported to the liver, alanine is reconverted to pyruvate and then to glucose. The glucose-alanine cycle (Figure 8.12) serves several purposes. In addition to its role in recycling ␣-keto acids between muscle and liver, the glucose-alanine cycle is a mechanism for transporting amino nitrogen to the liver. In ␣-keto acids, sometimes referred to as carbon skeletons, a carbonyl group is directly attached to the carboxyl group. Once alanine reaches the liver, it is reconverted to pyruvate. The amino nitrogen is then incorparated into urea or transfered to other ␣-keto acids to restore the amino acid balance in the liver (Chapter 15). A lanine transaminase P yruvate + O − CH ₃ O C C O L -Glutamate − O C CH ₂ CH ₂ C C O − O H O +NH ₃ + − O C CH ₂ CH ₂ C C O − O O O L -Alanine -Ketoglutarate C O − CH ₃ H C O +NH ₃ a

(26)

Gluconeogenesis Regulation

As with other metabolic pathways, the rate of gluconeogenesis is affected pri-marily by substrate availability, allosteric effectors, and hormones. Not surpris-ingly, gluconeogenesis is stimulated by high concentrations of lactate, glycerol, and amino acids. A high-fat diet, starvation, and prolonged fasting make large quantities of these molecules available.

The four key enzymes in gluconeogenesis (pyruvate carboxylase, PEP car-boxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase) are affected to varying degrees by allosteric modulators. For example, fructose-1,6-bisphos-phatase is activated by citrate and inhibited by AMP and fructose-2,6-bisphos-phate. Acetyl-CoA activates pyruvate carboxylase. (The concentration of acetyl-CoA, a product of fatty acid degradation, is especially high during star-vation.) Figure 8.13 provide an overview of the allosteric regulation of glycoly-sis and gluconeogeneglycoly-sis.

FIGURE 8.13

Allosteric Regulation of Glycolysis and Gluconeogenesis

The key enzymes in glycolysis and gluconeogenesis are regulated by allosteric effectors. Activator, ; inhibitor, .

Hexokinase Glucose Glucose-6-phosphate Glucose-6-phosphatase Fructose-6-phosphate Fructose-1,6-bisphosphatase (+) Citrate (−) bisphosphate (−) AMP Phospofructokinase (+) bisphosphate (+) AMP (−) ATP (−) Citrate Fructose-1,6-bisphosphate Phosphoenolpyruvate Phosphoenolpyruvate Carboxykinase Pyruvate Oxaloacetate (−) phosphate Pyruvate carboxylase (+) Acetyl-CoA Pyruvate kinase (−) ATP (−) bisphosphate (−) Acetyl-CoA (−) cAMP-dependent phosphorylation

(27)

8.3 The Pentose Phosphate Pathway 27

As with other biochemical pathways, hormones affect gluconeogenesis by altering the concentrations of allosteric effectors and the key rate-determining enzymes. As mentioned previously, glucagon depresses the synthesis of fructose-2,6-bisphosphate, which releases the inhibition of fructose-1,6-bisphosphatase, and inactivates the glycolytic enzyme pyruvate kinase. Hormones also influence gluconeogenesis by altering enzyme synthesis. For example, the synthesis of glu-coneogenic enzymes is stimulated by cortisol, a steroid hormone produced in the cortex of the adrenal gland that facilitates the body’s adaptation to stressful situations. Finally, insulin action leads to the synthesis of new molecules of glu-cokinase, PFK-1 (SREBP1c-induced), and PFK-2 (glycolysis favored). Insulin also depresses the synthesis (also via SREBP1c) of PEP carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase. Glucagon action leads to the synthesis of additional molecules of PEP carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase (gluconeogenesis favored).

The hormones that regulate glycolyses and gluconeogenesis alter the phos-phorylation state of certain target proteins in the liver cell, which in turn mod-ifies gene expression. The key point to remember is that insulin and glucagon have opposing effects on carbohydrate metabolism. The direction of metabolite flux, (i.e., whether either glycolysis or gluconeogenesis is active) is largely determined by the ratio of insulin to glucagon. After a carbohydrate meal, the insulin/glucagon ratio is high and glycolysis in the liver predominates over gluconeogenesis. After a period of fasting or following a high-fat, low-carbohydrate meal, the insulin/glucagon ratio is low and gluconeogenesis in the liver predominates over glycolysis. The availability of ATP is the second important regulator in the recip-rocal control of glycolysis and gluconeogenesis in that high levels of AMP, the low-energy hydrolysis product of ATP, increase the flux through glycolysis at the expense of gluconeogenesis, and low levels of AMP increase the flux through gluconeogenesis at the expense of glycolysis. Although control at the PFK-1/fructose-1,6-bisphosphatase cycle would appear to be sufficient for this pathway, control at the pyruvate kinase step is key because it permits the maximal retention of PEP, a molecule with a very high phosphate transfer potential.

8.3 THE PENTOSE PHOSPHATE PATHWAY

The pentose phosphate pathway is an alternative metabolic pathway for glucose oxidation in which no ATP is generated. Its principal products are NADPH, a reducing agent required in several anabolic processes, and ribose-5-phosphate, a structural component of nucleotides and nucleic acids. The pentose phosphate pathway occurs in the cytoplasm in two phases: oxidative and nonoxidative. In the oxidative phase of the pathway, the conversion of glucose-6-phosphate to ribulose-5-phosphate is accompanied by the production of two molecules of NADPH. The nonoxidative phase involves the isomerization and condensation of a number of different sugar molecules. Three intermediates in this process that are useful in other pathways are ribose-5-phosphate, fructose-6-phosphate, and gly ceraldehyde-3-phosphate.

The oxidative phase of the pentose phosphate pathway consists of three reac-tions (Figure 8.14a). In the first reaction, glucose-6-phosphate dehydrogenase (G-6-PD) catalyzes the oxidation of glucose-6-phosphate. 6-Phosphogluconolactone and NADPH are products in this reaction. 6-Phospho-D-glucono-␦-lactone is then hydrolyzed to produce 6-phospho-D-gluconate. A second molecule of NADPH is produced during the oxidative decarboxylation of 6-phosphogluconate, a reaction that yields ribulose-5-phosphate.

A substantial amount of the NADPH required for reductive processes (i.e., lipid biosynthesis) is supplied by these reactions. For this reason this pathway is most active in cells in which relatively large amounts of lipids are synthesized, (e.g., adipose tissue, adrenal cortex, mammary glands, and the liver). NADPH is

K E Y C O N C E P T S

• Gluconeogenesis, the synthesis of new glucose molecules from noncarbohydrate precursors, occurs primarily in the liver. • The reaction sequence is the reverse of

glycolysis except for three reactions that bypass irreversible steps in glycolysis.

(28)

28 CHAPTER EIGHT Carbohydrate Metabolism OH HO O H O CH ₂O OH H OH H CH ₂O OH H OH H OH H CH ₂O O CH ₂ O H + CO ₂ H ₂O a-D-Glucose-6-phosphate Glucose-6-phosphate dehydrogenase

6-Phospho- D-glucono-d-lactone

6-Phospho- D-gluconate 3-Keto-6-phospho- D-gluconate D-Ribulose-5-phosphate 6-phosphogluconate dehydrogenase Gluconolactonase OH HO O H O CH ₂ HO O H + H + H + + + P 3 2 PO ₃2 PO ₃2 − PO ₃2 − C CO O − O C OH H C C C CH ₂OH OH C O H (a) C C CO O − H OH H O H C C C − − _O P 3 2 − _O + NADPH + NADPH NADP+ NADP+ FIGURE 8.14a

The Pentose Phosphate Pathway

(29)

8.3 The Pentose Phosphate Pathway 29 OH H C OH H C H HO C CH₂ OH H C OH H C HO C H OH H C OH H C CH2 OH H C OH H C OH H C CH₂ C O OH H C H C O OH H C OH H C H HO C OH H C OH H C H CH₂ OH H C D-Ribulose-5-phosphate D-Ribose-5-phosphate D-Glyceraldehyde-3-phosphate D-Fructose-6-phosphate D-Erythrose-4-phosphate D-Glyceraldehyde-3-phosphate (b) D-Sedoheptulose-7-phosphate D-Xylulose-5-phosphate Ribosephosphate isomerase Ribulose-5-phosphate 3-epimerase Transketolase Transaldolase Transketolase H C O CH₂ C O H C CH₂OH O CH₂ C CH₂OH O CH₂ CH₂OH C O CH₂ C CH₂OH O PO3 2− PO32− PO3 2− PO32− PO32− PO32− PO32− PO32−

also a powerful antioxidant. (Antioxidants are substances that prevent the oxida-tion of other molecules. Their roles in living processes are described in Chapter 10.) Consequently, the oxidative phase of the pentose phosphate pathway is also quite active in cells that are at high risk for oxidative damage, such as red blood cells.

The nonoxidative phase of the pathway begins with the conversion of ribulose-5-phosphate to ribose-ribulose-5-phosphate by ribulose-ribulose-5-phosphate isomerase or to xylu-lose-5-phosphate by ribuxylu-lose-5-phosphate epimerase. During the remaining reactions of the pathway (Figure 8.14b), transketolase and transaldolase catalyze the interconversions of trioses, pentoses, and hexoses. Transketolase is a TPP-requiring enzyme that transfers two-carbon units from a ketose to an aldose. (TPP, thiamine pyrophosphate, is the coenzyme form of thiamine, also known as vitamin

FIGURE 8.14b The Pentose Phosphate Pathway

(b) The nonoxidative phase. When cells require more NADPH than pentose phos-phates, the enzymes in the nonoxidative phase convert ribose-5-phosphate into the glycolytic intermediates fructose-6-phosphate and glyceraldehyde-3-phosphate.